The realization that non-coding transcripts are remarkably abundant and participate in the regulation of protein- coding genes has revolutionized the study of eukaryotic gene expression. Yet little is known about how non- coding RNAs are made and function. We discovered an RNA polymerase II (Pol II) termination pathway that influences the synthesis and function many non-coding RNAs. This pathway requires the conserved helicase Sen1 and a collection of RNA-binding proteins that recognize the termination signal in the nascent transcript. The Sen1 pathway is essential in the model eukaryote S. cerevisiae (brewer's yeast), and mutations in the human Sen1 gene (SETX) cause the degeneration of motor neurons, leading to forms of ataxia and amyotrophic lateral sclerosis (ALS). Remarkably, Sen1-dependent termination regulates some protein-coding genes by transcription attenuation, which was previously thought to be restricted to bacteria. We will determine the molecular mechanism of Sen1-dependent termination using both genetic and biochemical approaches. Our structure/function studies will focus on two key proteins in the pathway, Sen1 and Pol II, in which we have isolated mutations that result in terminator read-through. Regulation of the IMD2 gene will be examined in detail as a paradigm for NTP homeostasis by Sen1-dependent attenuation and alternative start site selection. We will also identify and characterize new examples of Sen1-dependent gene regulation. The cis- and trans-acting determinants for regulation of these genes will be defined using genetic selections, quantitative gene expression assays, and mathematical modeling. The information gained in these studies will serve as a foundation for the investigation of RNA-based transcriptional regulation in other eukaryotes, including humans. Given the emerging evidence for the importance of non-coding transcripts in gene regulation in humans, our results will have broad implications for eukaryotic biology and human health, and will lead to a better understanding of the causes of motor neuron degeneration. PUBLIC HEALTH RELEVANCE: Mutations in the human Senataxin gene cause motor neuron degeneration that results in crippling muscle weakness, typically starting in the teen years. We discovered that the brewer's yeast homolog of Senataxin, called Sen1, has a crucial function in the regulation of gene expression. We will use yeast as model system to explore Sen1 function and better understand how defects in the human Senataxin protein lead to disabling diseases.